Streptococcus uberis adhesion molecule

ABSTRACT

A polypeptide, designated as “ Streptococcus uberis  Adhesion Molecule” (SUAM), and fragments of SUAM, prevent internalization and adherence of  Streptococcus uberis  and other streptococcal pathogens to cells. The SUAM polypeptide and fragments may be used diagnostically and therapeutically. Nucleic acid sequences encoding the SUAM polypeptide and fragments are included in the invention.

The invention was developed in part by a research grant from the UnitedStates Department of Agriculture and the U.S. government may thereforehave certain rights to the invention.

This application claims the priority of U.S. patent application Ser. No.10/691,384, filed on Oct. 22, 2003, which issued as U.S. Pat. No.7,517,955 on Apr. 14, 2009, which claims priority from U.S. ProvisionalPatent Application Ser. No. 60/429,499, filed on Nov. 26, 2002.

FIELD OF THE INVENTION

The invention pertains generally to the field of antigenic proteins andpolypeptides.

Specifically, the invention pertains to the field of polypeptides thatare useful to diagnose the presence of an infection and to elicit animmune response against a bacterial pathogen, especially streptococcalpathogens.

BACKGROUND OF THE INVENTION

Streptococcus is a genus of bacteria that causes disease in humans andother animals.

In humans, one of the most important streptococcal pathogens isStreptococcus pyogenes, the causative organism of strep throat, scarletfever, and rheumatic fever. In cattle, streptococcal infections are asignificant cause of disease, such as mastitis.

Mastitis affects virtually every dairy farm and has been estimated toaffect 38% of all cows. The disease causes destruction ofmilk-synthesizing tissues which reduces milk production and alters milkcomposition. In severe cases, the productive performance of dairy cattlemay be diminished permanently. Thus, mastitis continues to be the singlegreatest impediment to profitable dairy production. Losses associatedwith mastitis cost American dairy producers about 2 billion dollars peryear and cost dairy producers worldwide an estimated 25 billion dollarsper year.

Current mastitis control programs devised in the 1960's are basedprimarily on hygiene including teat disinfection, antibiotic therapy andculling of chronically infected cows. Acceptance and application ofthese measures has led to considerable progress in controllingcontagious mastitis pathogens such as Streptococcus agalactiae andStaphylococcus aureus. However, postmilking teat disinfection andantibiotic dry cow therapy have been less effective againstenvironmental mastitis pathogens. Studies have shown that as theprevalence of contagious mastitis pathogens was reduced, the proportionof intramammary infections (IMI) by environmental pathogens increasedmarkedly.

Therefore, it is not surprising that environmental mastitis has become amajor problem in many well-managed dairy farms that have successfullycontrolled contagious pathogens. In these herds, environmentalstreptococci account for a significant number of both subclinical andclinical IMI in lactating and nonlactating cows. EnvironmentalStreptococcus species involved in bovine mastitis include Streptococcusuberis, Streptococcus dysgalactiae subsp. dysgalactiae, Streptococcusequinus (formerly referred to as Streptococcus bovis), Streptococcusequi, Streptococcus parauberis and Streptococcus canis. Among theenvironmental streptococci, S. uberis and S. dysgalactiae subsp.dysgalactiae appear to be the most prevalent, infecting mammary glandsas favorable conditions arise.

In spite of the economic impact caused by the high prevalence ofenvironmental streptococci in many well-managed dairy herds, virulencefactors associated with pathogenesis of environmental streptococcalmastitis in dairy cows are not well understood. This constitutes a majorobstacle for development of strategies to control these importantmastitis pathogens. Consequently, strategies for controlling mastitiscaused by environmental streptococci are poorly defined and currentlyinadequate.

A significant need exists for effective therapies to combatstreptococcal infections, both in domestic animals and in people, andfor effective modalities by which the presence of a streptococcalinfection may be definitively diagnosed.

Survival of pathogenic microorganisms, such as Streptococci, hasdepended on the evolution of a range of strategies for evasion of hostdefenses. Associated with this evolution is the expression of a varietyof virulence determinants that favor persistence of bacteria in the faceof a massive inflammatory cell infiltration. In the case of bovinemastitis, it is hypothesized that adherence to and subsequentinternalization of mastitis pathogens into mammary epithelial cells isan important early event in the establishment of new intramammaryinfections in lactating and nonlactating mammary glands of dairy cows.Virulence factors that favor adherence and internalization to host cellsplay a crucial role in the establishment, spread, and persistence ofinfection. During the last decade, research from our laboratory hasfocused extensively on development of in vivo and in vitro models tostudy host-pathogen interactions, and especially on identification andcharacterization of virulence factors associated with the pathogenesisof S. uberis mastitis.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A-1D is a series of bar graphs showing the effects of antibodiesdirected against SUAM (A and B) and pepSUAM (C and D) on adherence andinternalization of S. uberis into bovine mammary epithelial cells.

FIG. 2 is a diagrammatic representation of a proposed lactoferrin bridgemodel for adherence of Streptococcus uberis to bovine mammary epithelialcells.

FIG. 3 is the theoretically elucidated SUAM gene sequence. (Seq. ID No.1)

FIG. 4 shows the translation of the nucleotide sequence of Seq. ID No. 1in the correct reading frame. (Seq. ID No. 2)

FIG. 5 is the DNA sequence of the SUAM gene. (Seq. ID No. 3)

FIG. 6 shows the translation of the nucleotide sequence of Seq. ID No. 3in the correct reading frame. (Seq. ID No. 13 to Seq. No. 17)

DESCRIPTION OF THE INVENTION

In this application, the terms “Streptococcus uberis Adhesion Molecule”or “SUAM” is preferably used although the terms “StreptococcusLactoferrin-binding Protein”, “Lactoferrin Binding Protein” and “LBP”are also used to refer to the same polypeptide. The terms “Streptococcusuberis Adhesion Molecule” and “SUAM” are preferred so as not to confusethe polypeptide of the present invention with the protein identified as“Streptococcus uberis Lactoferrin-Binding Protein” in Jiang et al., WO98/21231. The Jiang protein is a different protein than the SUAM of thepresent invention. Protein-nucleic acid TBLASTN (National Center forBiotechnology Information) and Swissprot amino acid data bank were usedto align the SUAM N-terminal amino acid sequence with previouslysequenced genes and proteins including S. uberis LBP described by Jianget al. No similarities were found, thus indicating that the SUAMbacterial protein of the invention is novel.

Recently, it has been shown that S. uberis binds to purified bovine milklactoferrin (LF) and that at least two proteins from S. uberis wereinvolved in this binding. Fang and Oliver, FEMS Microbiol. Lett., 176:91(1999). It has further been shown that LF appears to function as abridging molecule between S. uberis and bovine mammary epithelial cells,facilitating adherence of this mastitis pathogen to host cells. Fang, etal., American Journal of Veterinary Research, 61:275 (2000). Thisresearch indicates that the S. uberis proteins that bind to LF influenceadherence of S. uberis to mammary epithelial cells and internalizationof S. uberis into bovine mammary epithelial cells.

Further research in our laboratory has provided the followingdiscoveries.

(1) A 112 kDA protein from S. uberis that binds to LF was isolated andpurified and an N-terminal amino acid sequence of this 112 kDa proteinwas determined. The sequence is that of a novel protein, which isreferred to herein as Streptococcus uberis Adhesion Molecule or SUAM.

(2) SUAM-like proteins were identified in other Streptococci, includingStreptococcus dysgalactiae subsp. dysgalactiae and Streptococcusagalactiae.

(3) The SUAM-like proteins produced by S. dysgalactiae subsp.dysgalactiae bind to bovine LF in a manner similar to that which occurswith S. uberis.

(4) Antibodies against SUAM (whole protein) and to a synthetic peptide(pepSUAM) encompassing 15 amino acids near the N-terminus of SUAM havebeen produced.

(5) These antibodies cross-react with homologous proteins present inother strains of S. uberis demonstrating that SUAM was produced by allstrains of S. uberis evaluated.

(6) Anti-pepSUAM and anti-SUAM antibodies cross-react with otherstreptococcal pathogens, including S. agalactiae, S. dysgalactiae subsp.dysgalactiae, and Streptococcus pyogenes.

(7) Antibodies directed against pepSUAM or SUAM inhibit adherence of S.uberis to, and internalization of S. uberis into, bovine mammaryepithelial cells. This establishes that pepSUAM and SUAM arebiologically active and are involved in adherence to and internalizationof S. uberis into bovine mammary epithelial cells, indicating theimportance of SUAM as a significant S. uberis virulence factor.

(8) A theoretical DNA sequence of SUAM was determined and confirmed byPCR and restriction digests.

(9) The “true” DNA sequence encoding for SUAM was elucidated and foundto have 99% homology to the theoretically elucidated SUAM DNA.

It is conceived that this single virulence factor (SUAM) plays acritical role in the pathogenesis of streptococcal mastitis byfacilitating bacterial adherence to bovine mammary epithelial cells. Itis conceived that S. uberis expresses SUAM and uses LF in milk and/or onthe epithelial cell surface to adhere to mammary epithelial cells. It isfurther conceived that antibodies that bind to SUAM or pepSUAM may beused to diagnose infections due to S. uberis or other streptococci or totreat infections due to S. uberis or other streptococci. It is furtherconceived that nucleic acid sequences that encode SUAM or pepSUAM may beused diagnostically or in the production of anti-streptococcal vaccines.It is further conceived that the SUAM and pepSUAM polypeptides of theinvention may be used to in the production of antisera or vaccines tocombat diseases due to S. uberis or other streptococci.

In one embodiment, the invention is a polypeptide comprising an aminoacid sequence of at least 6 sequential amino acids of pepSUAM(MTTADQSPKLQGEEA), designated herein as Seq. ID No. 4, wherein anantibody that binds to the polypeptide inhibits adherence to and/orinternalization of S. uberis into bovine mammary epithelial cells. Forexample the 6 sequential amino acids of the polypeptide of the inventionmay be amino acids 1 to 6, 2 to 7, 3 to 8, 4 to 9, 5 to 10, 6 to 11, 7to 12, 8 to 13, 9 to 14, or 10 to 15 of Seq. ID No. 4 pepSUAM.

Preferably, the polypeptide of this embodiment of the inventioncomprises an amino acid sequence of more than 6 sequential amino acidsof pepSUAM of Seq. ID No. 4, for example, 7, 8, 9, 10, 11, 12, 13, 14sequential amino acids, or the entire 15 amino acid sequence of Seq. IDNo. 4. The polypeptide of the invention may further contain additionalamino acids to the amino terminal or carboxy terminal sides of thesequence that is a portion or all of pepSUAM. For example, thepolypeptide of the invention may contain at its amino terminal end theamino acids DD, which are present at the amino terminal end offull-length SUAM.

The polypeptide may be used to elicit antibodies which may be used todiagnose infections due to SUAM-expressing organisms such asStreptococcus, like S. uberis. The polypeptide may also be used toelicit an immune response in an animal or human that is susceptible toinfection by an organism that contains a surface antigen that will bindto an antibody that binds to the polypeptide of the invention. Thus, thepolypeptide of the invention may be useful as a vaccine againstinfection due to Streptococcus, such as S. uberis, S. pyogenes, S.agalactiae, or S. dysgalactiae.

In another embodiment, the invention is an isolated SUAM proteinpreferably having the amino acid sequence shown in FIG. 4 or FIG. 6 anddesignated herein as Seq. ID No. 2 or Seq. ID No. 15, respectively.

In another embodiment, the invention is a polypeptide derived from SUAMprotein, which may be isolated by the method described below and whichcomprises the sequence of amino acids MTTADQSPKLQGEEA, Seq. ID No. 4.

The invention also includes polypeptides that are substantiallyhomologous with the pepSUAM polypeptide or SUAM protein and polypeptidesderived therefrom, as described above. As used in this context, the term“substantially homologous” means that the amino acid sequence shares atleast 50%, such as at least 60%, preferably at least 70%, morepreferably at least 80%, and most preferably at least 90% amino acididentity with the pepSUAM or SUAM protein or polypeptides derivedtherefrom and wherein an antibody that binds to the polypeptide inhibitsthe adherence and/or the internalization of S. uberis to bovine mammaryepithelial cells.

In another embodiment, the invention is an antibody that selectivelybinds to an amino acid sequence of any 6 to 15 sequential amino acids ofpepSUAM, as described above. Preferably, the antibody inhibits theadherence and/or the internalization of S. uberis to bovine mammaryepithelial cells. The antibody may be a monoclonal or polyclonalantibody and may be used diagnostically or therapeutically.

In another embodiment, the invention is an antibody that selectivelybinds to the SUAM polypeptides or proteins of the invention. Preferably,the antibody inhibits the adherence and/or the internalization of S.uberis to bovine mammary epithelial cells. The antibody may be amonoclonal or polyclonal antibody and may be used diagnostically ortherapeutically.

In another embodiment, the invention is an isolated nucleic acidsequence that encodes the pepSUAM polypeptide. Preferably, the nucleicacid sequence comprises the sequence shown in underline and in bold inFIG. 3, and designated Seq. ID No. 5:ATGACAACTGCTGATCAATCACCTAAATTACAAGGTGAAGAAGCA.

In another embodiment, the invention is an isolated nucleic acidsequence that encodes the SUAM protein. Preferably, the nucleic acidsequence comprises either of the sequence shown in FIG. 3 or 5,designated Seq. ID No. 1 and Seq. ID No. 3, respectively. Morepreferably, the nucleic acid sequence comprises the sequence fromnucleotide 317 to nucleotide 2836 of Seq. ID No. 1 or from nucleotide289 to nucleotide 2808 of Seq. ID No. 3. Most preferably, the nucleicacid sequence comprises the sequence from nucleotide 311 to nucleotide2836 of Seq. ID No. 1 or nucleotide 283 to nucleotide 2808 of Seq. IDNo. 3.

Also included in the isolated nucleic acid sequences of the invention isa nucleic acid sequence that will hybridize under highly stringentconditions, for example at 3×SSC at 65° C. and preferably at 6×SSC at65° C., to the complement of the above specifically described nucleicacid sequences.

In another embodiment, the invention is a method for immunizing ananimal or human with an antigen against a bacterial organism. Inaccordance with the method of the invention, the polypeptide of theinvention or the SUAM polypeptide is administered to an animal or humansubject by any suitable means such as by injection or intramammaryinfusion and the subject is thereby caused to produce antibodies thatselectively bind thereto, which antibodies inhibit bacteria that bind tolactoferrin from adhering and/or internalizing to cells and/or enhanceclearance of bacterial pathogens. In this way, the ability of themicroorganism to cause disease is reduced.

In another embodiment, the invention is a primer selected from the groupof

(Seq. ID No 6) (a) 5′-GTC ATT TGG TAG GAG TGG CTG-3′, (Seq. ID No 7) (b)5′-TGG TTG ATA TAG CAC TTG GTG AC-3′, (Seq. ID No 8) (c) 5′-GGA TGA CATGAC AAC TGC TGA TC-3′, (Seq. ID No 9) (d) 5′-CAA TTG TCA GCA CGT CTC TGTAC-3′, (Seq. ID No 10) (e) 5′-CTT GGA ACT GGT GTT GGT ATG G-3′, and(Seq. ID No 11) (f) 5′-CAG GTG TTA CTT CAG GTG CTA C-3′.

Preferably, the primers are grouped in pairs with primers (a) and (b)being paired as a forward and reverse PCR primer, respectively, primers(c) and (d) being paired as a forward and reverse PCR primer,respectively, and primers (e) and (f) being paired as a forward andreverse PCR primer, respectively.

The primers and primer pairs of the invention are useful, for example,in identifying microorganisms that produce SUAM or a polypeptidemolecule having a high degree of homology to SUAM, such as 70% or morehomology. As such, the primers of the invention may be used to diagnosethe presence of an infection with a SUAM polypeptide, or SUAM-likepolypeptide, producing microorganism. It is conceived that an animal orhuman patient that is diagnosed in this manner may be treated withadministration of the polypeptide of the invention to induce an immuneresponse against such microorganism.

Following is a list of possible applications of various embodiments ofthe invention.

This list is not intended to be all inclusive as those skilled in theart will understand that additional uses exist for the invention.

I. Antibodies to SUAM and pepSUAM

-   A. Commercial Use-   Diagnostic-   Microbiology: immuno-fluorescence, card-test for preliminary    confirmation (including cow-side rapid tests using milk from cows    with mastitis)-   Serology: Agglutination/precipitation tests (cow-side rapid tests),    ELISA Diagnostic enrichment of bacteria from crude samples-   Treatment/Prevention-   Therapy for cows with mastitis (systemic/intramammary)-   Prevention for new cows introduced to a farm with history of S.    uberis infection-   Intramammary preparations for cows near parturition    B. Research Use-   1. Isolation/purification of SUAM-   2. In vitro pathogenicity assays-   3. Recombinant protein expression (monitoring and isolation)-   4. Mutant detection-   5. Immuno-histochemistry-   6. Western blot-   7. Immunoprecipitation for protein/protein interaction studies-   8. Steric inhibition studies    II. SUAM Protein    A. Commercial Use-   1. Vaccine production-   2. Antisera production-   3. Protein as antigen component of multivalent vaccine    B. Research Use-   1. Antisera production-   2. Experimental vaccination studies-   3. Protein as antigen component of multivalent vaccine-   4. Protein as ligand in affinity purification of bovine lactoferrin    III. pepSUAM    A. Commercial Use-   1. Vaccine production-   2. Antisera production-   3. Peptide as antigen component of multivalent vaccine-   4. Peptide as competitive inhibitor of adhesion/invasion in    intramammary preps    B. Research Use-   1. Antisera production-   2. Experimental vaccination studies-   3. Peptide as component of multivalent vaccine-   4. Peptide as ligand in affinity purification of Bovine Lactoferrin    IV. SUAM DNA Sequence    A. Commercial Use-   Diagnostic-   1. Probes-   2. PCR (alternative primers design)-   3. Cow-side rapid test (i.e., cantilever)    Prevention of Mastitis-   1. Recombinant expression for vaccine production (baculo-virus    cloning and expression)-   2. DNA vaccines (cloning into retro-virus vectors or Agrobacterium    tumefaciens)-   3. Cloning and expression in vitro for vaccine production    B. Research Use-   1. Probes-   2. PCR (alternative primers design)-   3. Real time PCR for selection and identification of strains-   4. DNA microarrays/differential display (to identify and study    factors that enhance or repress SUAM expression)-   5. Site directed mutagenesis-   6. Production of avirulent carrier strain for this or any other    expressed protein vaccine.    V. SUAM PCR Primers    A. Commercial Use    Diagnostic-   1. PCR amplification products detected by any means-   2. Real time PCR (taq-man, beacons, etc.)-   3. Probes    B. Research Use-   1. PCR Detection-   2. Real time PCR (taq-man, beacons, etc.)-   3. Probes for southerns, reverse transcriptase protection assays,    etc.-   4. Cloning and expression

The invention is further illustrated in the following non-limitingexamples.

EXAMPLE 1 Identification of Streptococcus Uberis Lactoferrin-bindingProteins (Prior Art), (Described in Fang, W., and S. P. Oliver, FEMSMicrobiol. Lett. 176:91) (1999)

Experiments were conducted to examine binding of lactoferrin (LF) bystrains of S. uberis causing bovine mastitis and to identify proteinsfrom the bacteria involved in LF-binding. Four strains of S. uterisisolated originally from dairy cows with mastitis and S. uberisATCC13387 (American Type Culture Collection, Manassas, Va.) wereevaluated. After growth, bacterial cultures were washed and split intotwo equal portions: one for incubation in milk and the other inphosphate buffered saline (PBS) (as controls). Bacterial surfaceproteins from pellets were extracted using 0.2% sodium dodecyl sulfate(SDS) and electrophoresed. Gels were silver-stained or transferred ontonitrocellulose membranes for immunoblotting using rabbit anti-bovine LFantibody and HRP (horseradish peroxidase)-conjugated donkey anti-rabbitIgG antibody as probes.

Biotin-avidin-based binding assay (BABA) and ELISA-based binding assaywere carried out on immobilized S. uberis microplates. LF from bovinemilk and transferrin (TF) from bovine plasma were biotinylated. For theBABA assay, serial 2-fold dilutions of biotinylated LF were added intomicroplate wells. incubated, washed, and probed with HRP-NEUTRAVIDIN.The ELISA-based assay was essentially the same as BABA except thatserial 2-fold dilutions of unlabelled LF were substituted forbiotinylated LF. Rabbit anti-bovine LF antibody and HRP-conjugateddonkey anti-rabbit IgG antibody were used as probes. Inhibition ofI.F-binding by unlabelled LF, TF, mannose, galactose, and lactose werealso tested using BABA and ELISA.

Polypeptides that bound to LF were identified by SDS-PAGE and westernblot analysis of bacterial surface proteins. Blots were probedsequentially with LF, rabbit anti-bovine LF antibody, and HRP-conjugateddonkey anti-rabbit IgG antibody. At least two proteins in each strain ofS. uberis were identified as lactoferrin-binding proteins. Theseincluded 52 and ˜112 kDa bands in 4 of 5 strains evaluated. One strainof S. uberis did not have the 112 kDa protein band, however, this strainproduced a higher molecular weight protein (134 kDa) which also bound toLF.

The microplate-based assay systems demonstrated that S. uberis bound topurified LF. These studies provided evidence that S. uberis binds to LFin milk and that at least two proteins from S. uberis surface moleculesare involved in LF-binding.

Example 2 Effect of Lactoferrin on Adherence of Streptococcus Uberis toBovine Mammary epithelial cells (prior art) (described in Fang, W., R.A. Almeida, and S. P. Oliver, Am. J. Vet. Res. 61:275) (2000)

A series of experiments were conducted to determine effects of LF onadherence of S. uberis to mammary epithelial cells. Three strains of S.uberis were used. In the first experiment, we investigated the effect ofLF on adherence of S. uberis to bovine mammary epithelial cells. SterileLF in Dulbecco's Modified Eagle Medium (DMEM) or milk (0.5 ml) and 0.5ml of bacterial suspension containing 1-2×10⁸ cfu/ml in DMEM were addedto bovine mammary epithelial cell line (MAC-T) monolayers. Finalconcentrations for LF were 0, 0.01, 0.1 and 1 mg/ml. Those for milk were0, 12.5%, 25% and 50%. Bacteria were allowed to adhere to MAC-T cellsand supernatants were then aspirated and diluted for bacterial counting.Monolayers were washed and lysed, and cell lysates were 10-fold dilutedfor bacterial counting. Streptococcus uberis cultures were alsopretreated with LF (1 mg/ml) or milk (100%) for 1 h. Bacterialsuspensions in PBS (without LF or milk) were included as controls.Bacteria were then washed and adjusted to 1-2×10⁸ cfu/ml for adherenceassays.

To test the effect of anti-bovine LF antibody on adherence, a S. uberisstrain was pretreated with LF or milk as described above and examinedfor its adherence to MAC-T cells in presence of different dilutions ofrabbit anti-bovine LF antibody. For the microscopic adherence assay,strains of S. uberis were labeled with fluorescein isothiocyanate(FITC). FITC-labeled bacteria were resuspended in DMEM. Sterile LF inDMEM (0.15 ml) and FITC labeled bacteria (0.15 ml) were added to wellsof chamber slides containing confluent MAC-T cell monolayers. Afterincubation, bacterial supernatants were removed, and slides were washedand examined microscopically.

All strains of S. uberis evaluated bound to LF in milk and to purifiedLF. LF and milk enhanced adherence of S. uberis to MAC-T cells whenpresent in the test medium (P<0.05-0.01) except for one strain of S.uberis. Pretreatment of bacteria with LF and milk increased adherence ofone strain of S. uberis (P<0.01), but not the other two strains. It isconceived that differences between LF or milk pretreatment and presenceof LF or milk in the medium could partially account for the differentresults. Because LF is synthesized and secreted by mammary epithelialcells and also binds to mammary epithelial cells, it is conceived thatthe presence of LF in the test medium might enhance the potential of LFas a bridging molecule between bacteria and MAC-T cells, thus increasingadherence. Additionally, differences of intrinsic surface propertiesamong S. uberis strains might affect their interaction with LF as wellas with MAC-T cells. There were differences among these S. uberisstrains in hydrophobicity. Two strains of S. uberis were more attractedto hexadecane as well as to MAC-T cells than was a third strain of S.uberis.

The involvement of milk in the adherence of S. uberis to MAC-T cells maybe more complicated than that of purified LF because of the coexistenceof other milk components that may also play a part in bacterialinteractions with epithelial cells. For example, our laboratorydemonstrated and reported that adherence to extracellular matrixproteins, particularly collagen, enhanced adherence and internalizationof S. uberis to bovine mammary epithelial cells and that presence ofthese host proteins up-regulated expression of ligands for collagen.Therefore, LF is not the only host protein that binds to S. uberis.However, our data indicate specific involvement of LF in adherence sinceaddition of rabbit anti-bovine LF antibody significantly decreasedadherence of LF or milk-pretreated bacteria to MAC-T cells (P<0.01) atdilutions below 1:500 for LF and 1:100 for milk.

The results of these studies indicate that LF functions as a bridgingmolecule between S. uberis and bovine mammary epithelial cells andfacilitates adherence of this mastitis pathogen to the host cells.

EXAMPLE 3 Investigation of Influence of Strain of S. uberis on theEnhancing Effect of LF on Adherence and Internalization to MammaryEpithelial Cells

To further investigate a possible strain influence on the enhancingeffect of LF on adherence and internalization to mammary epithelialcells, additional studies were conducted. In these studies, six strainsof S. uberis isolated originally from milk of dairy cows with mastitiswere used. Bacteria were pretreated with LF (ICN, Aurora, Ohio), 21.4%iron saturation and 97.5% protein content, 1 mg/ml) for 1 h at 37° C.,washed 3 times with PBS (pH 7.4), resuspended in DMEM and coculturedwith MAC-T cells for 1 h. After incubation, supernatants were removed,monolayers were washed and either lysed with trypsin/triton solution todetermine total cell associated bacteria or treated with antibioticsolution to determine internalization of bacteria into mammaryepithelial cells. For the latter, after 2 h of incubation, antibioticsolution was removed, monolayers were washed 3 times with PBS and cellswere lysed with trypsin/triton solution. Colony forming units per ml(CFU/ml) in lysates were determined using standard colony countingtechniques. Although differences in adherence and internalization weredetected among strains, addition of LF caused significantly greateradherence or internalization to mammary epithelial cells of all strainsof S. uberis evaluated.

It is conceived that adherence and internalization are not two separateindependent events. Adherent bacteria are quickly internalized throughan endocytic-like mechanism, where receptors for the “bridging” proteinsare recycled and exposed in or on the host cell surface. The kinetics ofthese events has been described as a chain reaction where adherencepromotes internalization. Therefore, higher concentrations of the“bridging” protein results in increased adherence that in turn leads toincreased internalization rather that reversal of adherence. Thus,increased binding of LF by S. uberis mediated by a lactoferrin bindingprotein (SUAM) results in increased bacterial internalization intomammary epithelial cells.

Thus, it is conceived that, by a mechanism referred to herein as“molecular bridging” LF possesses different binding domains, a bindingdomain for the host cell and another binding domain for S. uberis (seethe schematic presented in FIG. 2 “Diagram 1”). The interaction betweenhost cell receptor and the host-domain region in S. uberis bound LFallows contact of the bacterium and host cell surface membrane resultingin adherence. The interaction between LF and its host cell receptortriggers arrangements on the host membrane that initiate theinternalization of the bacterium into the host cell.

EXAMPLE 4 Isolation, Purification and N-terminal Amino-acid Sequencingof Streptococcus uberis Adhesion Molecule (SUAM)

A study was conducted to compare potential differences in the efficiencyof extraction of SUAM with mutanolysin or SDS by SDS-PAGE and Westernblotting. Four strains of S. uberis were used. Bacterial surfaceproteins from cell pellets were extracted from 0.2% SDS in PBS (pH 7.2)following the method described by Fang and Oliver (1999). Each strain ofS. uberis was grown in THB (Todd-Hewitt Broth) (Difco Laboratories,Detroit, Mich.) at 37° C. overnight. After centrifugation, bacteria wereresuspended in PBS. Bacterial pellets were washed three times withsterile PBS, and surface proteins were extracted using 0.2% SDS (sodiumdodecyl sulfate) (Bio-Rad Laboratories, Hercules, Calif.; 30 mg wetweight of bacteria per 100 μl of 0.2% SDS) for 1 h at 37° C.

In the mutanolysin extraction method, a modified procedure was used.Bacterial cells were suspended (1 g/2 ml) in 50 mM phosphate buffer (pH7.2), containing 0.5 M sucrose and 10 mg/ml lysozyme (Sigma, St. Louis,Mo.). The resulting suspension was divided into 2 ml aliquots and 250units of mutanolysin (N-acetylmuramidase, Sigma) were added per aliquot.

The suspension was shaker incubated for 1 h at 37° C. Bacteria werepelleted by centrifugation and supernatants of each were removed andstored at −20° C.

Extracted bacterial surface proteins (10 μg/lane) were electrophoresedon 10% SDS-PAGE. Gels were stained with Coomassie brilliant blue ortransferred onto nitrocellulose membrane using Trans-Blot SD Semi-DryElectrophoretic Transfer Cell (Bio-Rad, Hercules, Calif.). Unbound siteson blots were blocked with 3% casitone. Blots were probed with LF (ICN,5 μg/ml) in PBS TWEEN 20 (PBST) containing 0.1% casitone for 6 h at 4°C. followed by four washes with PBST. Procedures for further probing ofblots with rabbit anti-bovine LF antibody and HRP-conjugated donkeyanti-rabbit IgG antibody were as described previously (Fang and Oliver,1999). Blots without probing with LF and rabbit anti-bovine LF antibodywere included as negative controls.

When surface proteins were extracted with 0.2% SDS detergent andevaluated by SDS-PAGE, 110 kDa and 112 kDa protein bands were extractedmore efficiently compared to the mutanolysin extraction method. InWestern blot analysis, the intensity of SUAM bands in SDS extracts,particularly 110 and 112 kDa, were much stronger than those ofmutanolysin extracts. Results of this study indicate that SDS extractsproteins of interest (110 kDa and 112 kDa) more efficiently and is apreferred method for SUAM purification and subsequent characterization.

EXAMPLE 5 Iron Availability Influences Expression of SUAM

A study was conducted in which the effect of an iron chelator onexpression of S. uberis was evaluated. Strains of S. uberis were growneither in THB or THB treated with the iron chelator 2,2-dipyridyl andsurface proteins from bacterial pellets were analyzed by Western blotusing LF as a probe and rabbit anti-bovine LF antibody. Western blotanalysis showed two major bands of 110 KDa and 112 KDa, respectively,with LF-binding activity.

In addition, LF-binding activity decreased in the presence of an ironchelator which indicates that iron in the medium influences expressionof SUAM.

EXAMPLE 6 Purification of SUAM

Thirty ml of PBS (pH 7.4) containing 30 mg of SDS-extracted S. uberissurface proteins were loaded into a bovine LF-coupled CNBr-activatedSEPHAROSE 4B column. SDS-extracted surface proteins were incubated withshaking for 2 h at 4° C. with 7 ml of SEPHAROSE 4B covalently linked tobovine LF (ICN, 21.4% iron saturation and 97.5% protein content). TheLF-SEPHAROSE 4B slurry was loaded into a chromatography column (1.25 cm×9 cm; total volume 70 ml) (Pfizer, New York, N.Y.). The column wassubsequently washed with 10 volumes of TBS (50 mM TRIS-HCl (pH 7.4) +150mM NaCl containing 0.1% TRITON X 100) to remove nonspecific-bindingproteins using a peristaltic pump at a flow rate of 1 ml/min untilabsorbance at 280 nm approached zero. The column was eluted with asodium chloride gradient from 0.1 M to 1 M NaCl in TBS. Fractions(10ml/fraction) were analyzed by absorbance at 280 nm, SDS-PAGE andWestern blot using LF, rabbit anti-bovine LF antibodies and biotinylatedLF as probes. Fractions containing SUAM were pooled, dialyed against PBSand stored at −70° C. until use.

Analysis of fractions revealed the presence of a protein in fractionnumber 14 to 32 eluted at 0.5 M NaCl. The molecular mass was estimatedto be ˜112 kDa using GEL SCAN (Corbett Research, Mortlake, NSW,Australia). Results from SDS-PAGE and Western blot analysis indicatedthat this band had LF-binding affinity. The yield of purified SUAM was20 Fg/ml (total 10 ml) from 3 liters of THB-grown S. uberis .

Example 7 N-Terminal Amino Acid Sequence of the 112 kDa SUAM

Excised PVDF membrane (PerkinElmer Life and Analytical Sciences, Inc.,Boston, Mass.) containing ˜ 112 kDa SUAM band was analyzed. The proteinwas sequenced on an Applied Biosystems model 477A sequencer (AppliedBiosystems, Foster City, Calif.) equipped with on-line PTH analysisusing the regular program O3RPTH. The PTH-derivatives were separated byreverse-phase HPLC over a BROWNLEE C-18 column (220 ×2.1 mm). Theinitial yield for the coupling step was calculated from the amount ofPTH-derivatives present in the first cycle and by the amount of proteinspotted. As a standard marker for amino acid sequence, the repetitiveyield from myoglobin was determined from peak heights of valine,leucine, and glutamic acid according to the positions. The repetitiveyield from β-lactoglobulin was calculated for leucine, isoleucine, andvaline residues. The N-terminal amino acid sequence of SUAM was D D M TT A D Q S P K L Q G E E A (T/A) L (I/A) (V/K) (Seq. ID No. 12).

The above procedures were repeated and an identical amino acid sequencewas obtained. A protein-nucleic acid TBLASTN search (NCBI) and Swissprotamino acid data bank search were used to align the SUAM-terminal aminoacid sequence with previously sequenced genes and proteins. Nosimilarities were found establishing that the bacterial SUAM protein isa novel protein.

EXAMPLE 8 Identification of Lactoferrin Binding Proteins inStreptococcus Dysgalactiae Subsp. Dysgalactiae and StreptococcusAgalactiae Isolated from Cows with Mastitis (Prior Art) (Described inPark, H. M., R. A. Almeida, and S. P. Oliver, FEMS Microbiol. Lett.207:87 (2000))

This paper demonstrates the presence of lactoferrin-binding proteins intwo major bovine mammary pathogens, Streptococcus dysgalactiae subsp.dysgalactiae (S. dysgalactiae) and Streptococcus agalactiae.

Three strains of S. dysgalactiae and five strains of S. agalactiae wereused to identify lactoferrin-binding proteins (LBPs). LBPs fromextracted surface proteins were detected by polyacrylamide gelelectrophoresis and Western blotting. All strains of S. dysgalactiaeevaluated had 52 kDa and 74 kDa protein bands. All strains of S.agalactiae evaluated had 52 kDa, 70 kDa and 110 kDa protein bands. Inaddition, a 45 kDa band was detected in two of five S. agalactiaestrains evaluated. This study demonstrated that S. dysgalactiae and S.agalactiae of bovine origin contain at least two major LBP's. Thus,LBP's are present in several Streptococcus species that cause mastitisin dairy cows.

EXAMPLE 9 Binding of bovine lactoferrin to Streptococcus dysgalactiaesubsp. dysgalactiae isolated form cows with mastitis (prior art) (Park,H. M., R. A. Almeida, D. A. Luther, and S. P. Oliver, FEMS Microbiol.Lett. 208:35 (2000))

Three strains of S. dysgalactiae subsp. dysgalactiae (one of which isstrain ATCC 27957) were used to determine if bovine lactoferrin (LF)binds to bacterial cells by biotin avidin binding assay (BABA),enzyme-linked immunosorbent assay (ELISA), and binding inhibition assay.Binding assays revealed that all strains of S. dysgalactiae subsp.dysgalactiae (S. dysgalactiae) evaluated in this study bound to LF,although some differences in LF binding capability among strains andbetween methods used were detected. Binding of LF was not inhibited bytransferrin (TF) and LF moiety molecules (mannose, galactose, andlactose) but by LF. This study demonstrates that S. dysgalactiae bindsto bovine LF in a specific manner.

EXAMPLE 10 Production of Antibodies Against SUAM (Whole Protein) and toa Synthetic Peptide (pepSUAM) Encompassing 15 Amino Acids Near theN-Terminus of SUAM

SUAM antibodies were needed to test the biological role of SUAM onadherence to and internalization of S. uberis into bovine mammaryepithelial cells, and to test protective effects of SUAM antibody onthese in vitro approaches. To obtain antibodies, purified SUAM asdescribed in Example 6 was sent to Quality Bioresources Inc. (QBI,Seguin, Tex.) for custom antibody production. For production ofantibodies against SUAM, ˜300 μg of purified protein was used toimmunize two rabbits. For production of antibodies against SUAM-derivedpeptide (pepSUAM), Bethyl Laboratories, Inc. (Montgomery, Tex.)synthesized the selected peptide based on the N-terminal amino acidsequence M T T A D Q S P K L Q G E E A (Seq. ID No. 4). All peptideswere HPLC purified and conjugated to KLH for immunization. PepSUAMinduced a high immune response with production of immunologic responsewhich yielded 20 mg of affinity purified antibody.

EXAMPLE 11 Cross-Reactivity of pepSUAM and SUAM Antibodies with SeveralStrains of S. Uberis.

To ensure that SUAM is not a rare protein found only in one strain of S.uberis, and that research or prophylactic products developed will havebroad significance, several strains of S. uberis from diverse locationswere tested by Western blotting. Strains evaluated were from Tennssee,Colorado, Washington and New Zealand. The different S. uberis strainswere cultured overnight in Todd Hewitt broth and surface proteins wereextracted in Laemmli sample buffer. SDS-PAGE polyacrylamide gels (7.5%)were electrophoresed followed by transfer to nitrocellulose membranes.They were blocked in PBSTG (phosphate buffered saline, 0.05% (v/v)TWEEN-20 , and 0.1% (w/v) porcine gelatin) for 1 h. Affinity purifiedrabbit anti-pepSUAM and rabbit anti-SUAM antibodies were diluted inPBSTG (1:2000) and blots treated for 1.5 h. Following washing of blotswith several changes of PBST, a 1:2000 dilution in PBSTG ofperoxidase-conjugated affipure F(ab′)2 fragment donkey anti-rabbit IgG(H+L) was applied. The SUAM protein band was revealed with theperoxidase substrate 4 CN (4-chloro-1-naphthol ). The presence of asingle dominant band on a blot of total S. uberis detergent extractedsurface proteins attests to the specificity of the antibodies. The 112kDa SUAM protein band is clearly visible. These results establish thatSUAM is a ubiquitous protein in S. uberis strains and that pepSUAM mayplay a role as a universal immunogen to protect against S. uberismastitis.

EXAMPLE 12 Cross-Reactivity of pepSUAM and SUAM Antibodies with S.Agalactiae, S. Dysgalactiae Subsp. Dysgalactiae, and StreptococcusPyogenes.

Cross-reactivity of rabbit anti-SUAM whole protein antibodies and rabbitanti-pepSUAM antibodies between different Streptococcus species wasinvestigated. Strains of S. dysgalactiae subsp. dysgalactiae, Sagalactiae (from animals and humans), and Streptococcus pyogenes werecultured overnight in Todd Hewitt broth and bacterial surface proteinswere extracted in Laemmli sample buffer. SDS-PAGE polyacrylamide gels(7.5%) were electrophoresed followed by transfer to nitrocellulosemembranes. They were blocked in PBSTG (phosphate buffered saline. 0.05%(v/v) TWEEN-20, and 0.1% (w/v) porcine gelatin) for 1 h. Affinitypurified rabbit anti-pepSUAM and rabbit anti-SUAM antibodies werediluted in PBSTG (1:2000) and blots treated for 1.5 h. The nexttreatment after washing blots with several changes of PBST was a 1:2000dilution in PBSTG of peroxidase-conjugated affipure F (ab′) 2 fragmentdonkey anti-rabbit IgG (H+L). The SUAM protein band was revealed withthe peroxidase substrate 4CN (4-chloro-1-naphthol ). Western blotresults showed cross reaction of pepSUAM and SUAM antibodies withproteins of other Streptococcus species, including the human pathogen S.pyogenes. The cross reaction with other proteins or protein fragmentsindicates that SUAM and its functions are conserved or partiallyconserved between Streptococcus species and that a vaccine based uponSUAM would have broad application.

EXAMPLE 13 Inhibitory Effect of SUAM and pepSUAM Antibodies on Adherenceand Internalization of S. uberis to Bovine Mammary Epithelial Cells

Two strains of S. uberis isolated from cows with clinical mastitis wereincubated with increasing concentrations of SUAM and pepSUAM antibodies,co-cultured with bovine mammary epithelial cells and adherence of S.uberis to and internalization of S. uberis into mammary epithelial cellsmeasured.

A bovine mammary epithelial cell line (MAC-T) was used. MAC-T cells werecultured in cell growth medium (CGM) in 24-well plates and incubated in5% CO₂/balance air at 37° C. Monolayers were checked daily forconfluence.

Two S. uberis strains isolated from cows with mastitis were used. Foradherence and internalization assays, bacteria stored at −70° C. werethawed in a 37° C. water bath, streaked onto blood agar plates, andincubated for 16 h at 37° C. Bacteria were then inoculated intoTodd-Hewitt broth (THB, Difco, Detroit, Mich.) for 2 h at 37° C.Bacterial suspensions were diluted in CGM to a concentration of 10⁷bacteria per ml.

Each of the two strains of S. uberis was preincubated with severaldilutions of SUAM and pepSUAM antibodies for 1 h at 37° C. Afterincubation, bacterial suspensions were washed three times to removeunbound antibodies and co-cultured with MAC-T cells for 2 h at 37° C. in5% CO2: 95% air (vol/vol). In order to enumerate bacteria associatedwith MAC-T cells (adherent+internalized bacteria), MAC-T cells werewashed 3 times to remove unbound bacteria and lysed with trypsin andtriton. MAC-T cell lysates were 10-fold serially diluted, seeded intriplicate on blood agar plates, and incubated overnight at 37° C. Afterincubation, individual colonies were counted and expressed as colonyforming units per ml (CFU/ml) of S. uberis.

In order to discriminate between S. uberis that adhered to the MAC-Tcell surface from those that were internalized into MAC-T cells, aninternalization assay was performed in parallel wells and under the sameculture conditions as described for the adherence assay. Theinternalization assay was similar to the adherence assay with theexception that an antibiotic treatment directed to destroy bacteria thatwere not internalized was performed before lysing MAC-T cells. Followingthis, MAC-T cells were washed extensively, lysed as described before,and bacteria that were internalized were enumerated as described for theadherence assay. The number of adherent bacteria was calculated bysubtracting the number of internalized bacteria from MAC-Tcell-associated bacteria.

Pretreatment with SUAM (FIGS. 1 A&B) or pepSUAM (FIGS. 1 C&D) antibodiesreduced adherence and internalization of S. uberis to mammary epithelialcells. The greatest adherence and internalization of S. uberis wasobserved when S. uberis was not pretreated with SUAM or pepSUAMantibodies. The lowest adherence and internalization of S. uberis wasdetected when higher concentrations of antibodies were used. FIG. 1 A-Dshow a dilution effect on adherence and internalization, which confirmsthe inhibitory effect of SUAM and pepSUAM antibodies on adherence to andinternalization of S. uberis into MAC-T cells. Results from thisexperiment showed the inhibitory effect of SUAM and pepSUAM antibodieson adherence and internalization of S. uberis into MAC-T cells andindicate the value of SUAM and pepSUAM as immunogens for controllingthis economically important disease of dairy cows.

EXAMPLE 14 Theoretical Elucidation of SUAM DNA Sequence and Confirmationby PCR and Restriction Digest

Theoretical elucidation of the DNA sequence from the pepSUAM amino acidsequence permitted DNA synthesis of the SUAM gene using PCR techniques.The pepSUAM amino acid sequence (MTTADQSPKLQGEEA) (Seq. ID No. 4) wasused to search a S. uberis genomic database (Wellcome Trust SangerInstitute) to identify a single fragment of the genome, also known as“contig”, that matched the DNA sequence of pepSUAM amino acids. Thematch for pepSUAM was 100% for this DNA contig and this was the onlymatch of this quality in the entire existing S. uberis genomic database.From this DNA contig, several PCR primers were designed and used in PCRreactions to obtain a unique DNA fragment. Subsequent analysis of thisPCR fragment showed physical and DNA sequence characteristics similar tothat of the elucidated SUAM gene. These results indicate that a uniqueand single gene of the S. uberis genomic sequence is responsible forcoding SUAM and that we generated unique PCR primers and defined PCRconditions for the synthesis of SUAM.

Using the ExPASy Home Page Translate Tool (Swiss Institute ofBioinformatics), the S. uberis genomic contig DNA sequence wastranslated to amino acid sequences, in all possible reading frames. Onlyone of the six possible translations contained an open reading frame (anarea without stop codons) long enough to code for the S. uberis protein.This sequence was checked using a BLAST search against the entireNational Center for Biotechnology Information (NCBI) genomic databaseand appears to be unique, with only partial segments showing homology.

The sequence shown in FIG. 3 is the hypothetical SUAM gene sequence withsome additional sequence included before and after, 3,041 nucleotides.This sequence is designated as Seq. ID No. 1. The coding region for theN-terminal sequence begins at nucleotide 311 and ends at 376(underlined). The coding region for the peptide used to generateantibody is from nucleotide 317 to 360 (bold). The open reading frame,i.e. gene, ends at the stop/termination codon represented by TAA,nucleotides 2837 to 2839.

FIG. 4 shows the translation of the nucleotide sequence of Seq. ID No. 1in the correct reading frame. This amino acid sequence is designatedSeq. ID No. 2. The N-terminal sequence segment is underlined and thepeptide used to generate the antibody to pepSUAM is underlined and bold.The end that corresponds to the above sequence (bold TAA in FIG. 3) ismarked by the dash following the bold GKK, which would be coded for byGGCAAAAAA.

This selected coding region was used to design primers for itsamplification by PCR. Three separate pairs of primers that bound to sixindividual sites were designed to generate three slightly differentfragments from this same gene. These primers successfully generated PCRproducts of the predicted length. This provides very strong evidencethat this gene is present in the strain of S. uberis (S. uberis UT888)from which the S. uberis protein (SUAM) was purified and the N-terminalpeptide sequence was determined. One of these primers was homologous tothe coding region for the N-terminal sequence providing further supportthat the correct gene was amplified.

In an effort to determine additional amino acid/protein and nucleicacid/DNA sequence, three independent pairs of PCR primers were designedfrom the S. uberis genomic database sequence, contig sub114a06.

TABLE 1 Name, nucleotide composition and expected PCR product size. SEQID PRODUCT NAME PRIMER NO. SIZE LFbpDL5forward 5′-GTC ATT TGG TAG GAGTGG CTG-3′ 6 2,970 bp LFbpDL6reverse 5′-TGG TTG ATA TAG CAC TTG GTGAC-3′ 7 2,970 bp LFbpDL7forward *5′-GGA TGA CAT GAC AAC TGC TGA TC-3′ 82,639 bp LFbpDL8reverse 5′-CAA TTG TCA GCA CGT CTC TGT AC-3′ 9 2,639 bpLFbpDL9forward 5′-CTT GGA ACT GGT GTT GGT ATG G-3′ 10 2,561 bpLFbpDL10reverse 5′-CAG GTG TTA CTT CAG GTG CTA C-3′ 11 2,561 bp *pepSUAMcoding region.PCR reaction was run using an iCycler (BioRad) and conditions used were:

-   Cycle 1: (1×) Step 1: 95° C. for 2 min-   Cycle 2: (30×) Step 1: 94° C. for 30 sec

Step 2: 94° C. for 30 sec

Step 3: 68° C. for 3 min

-   Cycle 3: (1×) Step 7: 68° C. for 7 min-   Cycle 4: (1×) Step 7: 4° C. holding    Reactions components used were as follows:-   Primer forward: 0.5 μM-   Primer reverse: 0.5 μM-   Genomic DNA template: 0.5 μg-   dNTP's: 200 μM each-   MgCl₂: 1.5 mM-   Taq polymerase: 0.825 U

PCR fragments obtained corresponded to the expected theoretical productsize (Table 1). These results indicate that the PCR fragments obtainedshow a high degree of similarity with the theoretical SUAM gene. Furtherconfirmation was done to compare the restriction enzyme map of the PCRfragments with the corresponding theoretical SUAM sequence.

Restriction Digest Confirmation: The longest PCR product of 2,970 bp,which includes a start and stop codon and therefore represents theentire gene, was further processed to confirm the specificity of the PCRreaction and further characterize the S. uberis SUAM gene. Restrictionenzyme digestion cuts DNA at specific locations that are recognized bythe different enzymes based upon their nucleotide sequence. The entiregene sequence was analyzed using NEBcutter at the New England BioLabsweb site. Three restriction enzymes, Bcl I, Hpa I, and Nla III werechosen based on their ability to recognize specific sequence sites thatwhen cut would generate distinctly identifiable fragments.

TABLE 2 Restriction enzymes, site of digestion (coordinates) andexpected length of digested DNA. Enzyme Coordinates (bp #) Length (bp)Bcl I  329-2632 2304 Bcl I 2633-3041 409 Bcl I  1-328 328 Hpa I1625-3041 1417 Hpa I   1-1204 1204 Hpa I 1205-1624 420 Nla III 1580-30411462 Nla III  320-1367 1048 Nla III  1-319 319 Nla III 1368-1579 212

Digestion of the 2,970 bp PCR fragment generated the expected patterns(lower molecular weight products were not clearly detected due todetection limits, as would be expected). The combined results of sixprimer binding sites and 10 restriction cut sites by 3 enzymes confirmedthat PCR fragments have a restriction pattern similar to that of thetheoretical SUAM sequence (FIG. 3, Seq. ID No. 1). These results,together with those from PCR reactions using several primercombinations, indicate that the PCR generated DNA fragment is similar tothe theoretical SUAM nucleic acid sequence.

EXAMPLE 15 DNA Sequencing of SUAM

The SUAM gene was amplified, cloned and sequenced from the mastitispathogen S. uberis strain UT 888. The results of this sequencing werethat S. uberis SUAM has 99% sequence identity to the theoretical SUAMgene identified in the Sanger S. uberis genomic database by homology tothe reverse translated peptide sequence described in Example 14.

The 2,970 bp PCR amplicon encompassing the SUAM gene was generated withprimers LFbpDL5forward and LFbpDL6reverse shown in Table 1 in Example 14(Seq ID Nos. 6 & 7). The product was gel purified from a 1.2% SeaPlaqueGTG agarose gel (BioWhittaker Molecular Applications, Rockland, Me.)with the QIAEX II gel extraction kit (Qiagen Inc., Valencia, Calif.).The cloning into plasmid pCR-XL-TOPO of the purified amplicon was by theinteraction of nontemplate-dependent polymerase generated adenine (A),overhangs of the amplicon and thymine (T), and overhangs of the vector.A mixture of recombinant Taq polymerase and Pyrococus DNA polymerase wasused to minimize polymerase reading error (Invitrogen, Carlsbad,Calif.). Chemically competent Escherichia coli, TOP 10 cells, weretransformed and selected on Luria-Bertani agar with 50 μg/ml kanamycin(Invitrogen, Carlsbad, Calif.). The positive clone was confirmed byisolation of the plasmid, (Wizard Plus SV miniprep DNA purificationsystem; Promega, Madison, Wis.), re-amplification of the insert, anddigestion with restriction enzymes (New England BioLabs, Inc., Beverly,Mass.) based upon restriction sites picked from the theoreticalsequence.

Confirmation of the theoretical sequence (The Wellcome Trust SangerInstitute, Hinxton, Cambs, UK) and determination of the actual sequencefrom S. uberis 888 was accomplished by automated DNA sequencing(Molecular Biology Resource Facility, The University of Tennessee,Knoxville, Tenn.) of the plasmid in the region of insertion in both aforward and reverse direction to sequence both strands. The firstprimers were of known sites on the plasmid; M13 forward and M13 reverse,with subsequent primers (Integrated DNA Technologies, Coralville, Iowa)being chosen from the 3′ end of the determined nucleic acid code. Fourrounds of sequencing yielded enough DNA sequence code to transverse theinsert in each direction. Sequence contig assembly was performed withthe aid of the software Sequencher ver. 4.0.2 (Gene Codes Corporation,Ann Arbor, Mich.).

As each forward and reverse contig was assembled, the overlappingregions provided a quality control check for sequencing error. When theforward and the reverse assembled contigs were compared, this providedan additional quality control check. There were at least two and oftenmore sequencing reactions used for each position in the final nucleicacid sequence. Final comparison and confirmation of the theoreticaldatabase sequence, and actual S. uberis UT 888 sequence were made withBLAST 2 SEQUENCES, BLASTN ver. 2.2.5 (National Center for BiotechnologyInformation, Bethesda, Md.). Results of this alignment were:Identities=2948 (theoretical)/2970 (actual) or 99% similarity.

The complete SUAM DNA sequence is presented in FIG. 5 and is designatedSeq. ID No. 5. The complete SUAM gene DNA sequence did not show homologywith other S. uberis genes reported in the Sanger S. uberis genomicdatabase. This indicates that the SUAM gene codes for a unique S. uberisprotein.

The amino acid sequence encoded by the SUAM DNA sequence of Seq. ID No.5 is presented in FIG. 6. Polypeptide fragments encoded by the DNASequence of Seq. ID No. 5 are shown in Seq. ID Nos. 13 to 17,respectively, in order of appearance in FIG. 6. In FIG. 6, the presenceof three sequential asterisks (***) indicates the position of a stopcodon in the nucleotide sequence of Seq. ID No. 5. The underlinedportion of amino acid sequence of FIG. 6 represents the N-terminalsequence of the SUAM protein. The underlined and bold portion of thesequence of FIG. 6 represents pepSUAM.

The SUAM polypeptide is shown in Seq. ID No. 15, preferably from aminoacids 64 to 905 and most preferably from amino acids 66 to 905. ThepepSUAM polypeptide is shown in Seq. ID No. 15 at amino acids 66 to 80.

The terms and expressions which have been employed in the foregoingspecification are used as terms of description and not limitation, andthere is no intention that the use of such terms and expressionsexcludes equivalents of the features shown and described above. Furthermodifications, uses, and applications of the invention described hereinwill be apparent to those skilled in the art. It is intended that suchmodifications be encompassed in the following claims.

1. An isolated nucleic acid that encodes a polypeptide comprising theamino acid sequence of SEQ ID NO:
 4. 2. The isolated nucleic acid ofclaim 1 that encodes a polypeptide comprising the amino acid sequence ofSEQ ID NO: 2 or SEQ ID NO:
 15. 3. The isolated nucleic acid of claim 1that comprises the nucleotide sequence of SEQ ID NO:
 5. 4. The isolatednucleic acid of claim 3 that comprises the sequence of nucleotides 317to 2836 of SEQ ID NO: 1 or the sequence of nucleotides 289 to 2808 ofSEQ ID NO: 3.